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Abstract

Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to distant cells, thereby affecting their gene expression, proliferation, differentiation, and function. Although their physiological functions are not clearly defined, recent studies have shown their therapeutic potential for tissue repair and regeneration. While MVs can be isolated readily from mesenchymal stem cells (MSCs) and other cell types from various sources, the yield of MVs under conventional culture condition in vitro is one of the limiting factors for both the in vivo functional study as well as in vitro molecular analysis. Here, we provide a protocol to increase the yield of microvesicles by preconditioning MSCs with rat brain extract.

Generation of neural stem cells or neural cells by direct reprogramming or utilization of mesenchymal stem cells for cell replacement therapy is potential options for neurodegenerative diseases (Adib et al., 2015). Recent studies have demonstrated that microvesicles derived from MSCs represent a novel and safe alternative to other cell replacement approaches to enhance tissue regeneration such as neuronal regeneration, immune modulation, angiogenesis in brain injury (Kim et al., 2013; Porro et al., 2015; Lee et al., 2016). Little is known about how external signals derived from damaged tissues can affect the quantity and composition of microvesicles. The contents and quantities of such functional secretome of MSCs can be significantly changed in response to their microenvironment (Qu et al., 2007). For example, ischemic brain extracts or hypoxia are known to induce the synthesis of a number of cytokines and growth factors that are beneficial to the tissue regeneration process (Chen et al., 2007; Shin et al., 2014). In the present study, normal and ischemic brain extracts as a form of brain injury signal were employed to increase the yields as well as to modulate the molecular composition of MVs from MSCs that can be beneficial for their clinical application. Indeed, the quantity of MVs in conditioned medium of MSCs was greatly increased by the treatment of normal brain extracts or ischemic brain extracts. The current protocol was mainly based on previously described methods (Choi et al., 2007; Kim et al., 2012) with a few modifications including reagents, recipes. The yield and composition of microvesicles can be significantly modulated by preconditioning of producing cells by physical, chemical or biological means. As an example, we utilized brain extract to stimulate MSCs to simulate signal for brain tissue damage and the final products (MVs) can be a potent specific therapy for brain tissue repair and regeneration. This protocol may provide a clue to develop better strategies to obtain higher yields of MVs with stronger therapeutic potential from various cell sources.

After the rats are perfused, use surgical scissors to remove the head with a cut posterior from the ears. Quickly make a midline incision in the head skin, aseptically remove the skull and meninges by bone rongeur.Note: The methods for rat sacrifice and brain tissue extraction were performed as previously described by Spijker (2011).

After placing whole brain on a brain matrix, make coronal dissection the region of the middle cerebral artery (bregma -1~+ 1 mm) on the ice from the ipsilateral hemisphere, collect the sliced brain tissue (Figure 1A).

Homogenize tissue pieces using tissue grinders.

Centrifuge tissue homogenates at 100,000 x g for 2 h at 4 °C, and then take supernatants.

Aspirate supernatant and resuspend the pellet in an appropriate volume of prewarmed complete medium.

Inoculate total of 50 flasks (75 cm2) with 20 ml/flask of 1 x 105 viable cells/ml for microvesicle isolation and return to incubator.Note: Cell cultures should be re-fed every 3 days with fresh complete medium for optimal cell growth.

Remove the caps from the feed and retentate ports of the Minimate TFF capsule.Note: Do not discard caps. They are required for storage.

Screw a male luer-to-hose-barb connector (included) into each of the feed/retentate ports.

Cut a piece of tubing 3.2 mm (1/8”) i.e., long enough to reach from the feed reservoir, through the pump head to the capsule.Note: Keep tubing lengths as short as possible to reduce system hold-up volume.

Connect the tubing to the hose-barb on one of the feed ports. Install the tubing in the pump head. Put the other end of the tubing into the reservoir.Notes:

If a pressure gauge or transducer is used, connect the tubing to the pressure device. Then connect the pressure device as close as possible to the feed port using suitable connectors.

Feed and retentate ports are interchangeable. Depending on the orientation of the capsule, choose the port that is at the lowest elevation as the feed port. This allows for air to be easily expelled when liquid is pumped through the capsule. The recommended crossflow for the Minimate TFF capsule is 30-40 ml/min.

Cut another piece of tubing, long enough to return from the retentate port to the sample reservoir.

Attach the tubing to the retentate hose-barb and put the other end in the reservoir. (Again, if a pressure gauge or transducer is used, the tubing connects to the pressure device, which must then be connected to the retentate port.)

Place the retentate screw clamp on the retentate tubing close to the retentate port (after the pressure gauge if installed). Secure in place but do not tighten to restrict the tubing.

Remove one of the filtrate caps.

Attach a female luer-to-hose-barb fitting to one of the filtrate/vent ports.

Note: This concentration ratio was selected based on the capacity of the subsequent ultracentrifugation steps. Thus, the concentration ratio may vary depending on the ultracentrifuge (rotor and tubes), cell types and culture condition employed.

After centrifugation, remove the supernatant (about 32 ml) carefully from sucrose fraction-enriched MVs layer. Note: MVs are about just above sucrose cushion (yellow line, Figure 4A) and should be handled with care because it is difficult to identify with the naked eyes (Figure 4B).

Figure 4. Enrichment of microvesicles by sucrose gradient ultracentrifugation. A. A discontinuous sucrose density gradient was prepared by layering 1.5 ml of 0.8 M sucrose gradient upon 0.5 ml of 2.7 M sucrose density solution. Next, 33 ml of concentrated conditioned medium was added to the top lay carefully. B. After centrifugation, microvesicles are enriched in the interface between medium and sucrose layers.

After centrifugation, identify a pellet at the bottom of the tube. Remove the PBS and dissolve the pellets with 100 μl of PBS.

The protein concentration of each fraction is determined using refractometer and the Bradford dye assay.Note: For therapeutic purposes, quantification of final products should be expressed as weight/ml (or unit/ml), if possible. Since protein is one of the main components of the microvesicles, protein quantification was employed. Alternatively, the quantification of MVs can be done by flow cytometry, nanoparticle tracking analysis (NTA) or resistive pulse sensing combined with Raman microspectroscopy.

Data analysis

The optimal time point for harvesting conditioned medium of hMSCs for later microvesicle isolation was determined based on the following assays:

To quantify the protein concentration in untreated-MVs and brain extract treated-MVs, samples were measured by Bradford method (Table 1).

Table 1. Protein amount in MVs from 1 L of conditioned MSCs medium

Notes

The quantity of the resulting microvesicles can be significantly varied depending on MSC tissue source, MSC cell density, passage number, duration of culture and culture microenvironment (biological, chemical or physical stimulations) for conditioned medium harvesting and the concentration of brain extract.
The processed data and statistical analyses are published in Lee et al. (2016), which can be found at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016792/

This protocol is adapted from ‘Microvesicles from brain-extract-treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke’ (Lee et al., 2016). This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP), No. 2012M3A9B4028639.